Increased proteome coverage by combining PAGE and peptide isoelectric focusing: comparative study of gel-based separation approaches.
Bottom Line: We have established a 3D workflow for shotgun proteomics that relies on protein separation by 1D PAGE, gel fractionation, trypsin digestion, and peptide separation by in-gel IEF, prior to RP-HPLC-MS/MS.Our results show that applying peptide IEF can significantly increase the number of proteins identified from PAGE subfractionation.This method delivers deeper proteome coverage and provides a large degree of flexibility in experimentally approaching highly complex mixtures by still relying on protein separation according to molecular weight in the first dimension.
Affiliation: Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.Show MeSH
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Mentions: After showing the high technical reproducibility of pIEF-LC-MS/MS after PAGE we set to examine the reproducibility of the combined PAGE-pIEF-LC-MS/MS approach. As this method relies heavily on PAGE separation in the first dimension, we performed two separate experiments using PAGE in which peptides extracted from a PAGE slice were either (i) analyzed directly by LC-MS/MS separation without pIEF (GeLC-MS/MS) or (ii) separated by pIEF prior to LC-MS/MS analysis (PAGE-pIEF-LC-MS/MS). In both experiments we used three parallel replicate PAGE lanes of 70 μg HeLa NE. PAGE lanes were fractioned, and for further analysis we selected the same set of three PAGE slices (PAGE#05, PAGE#10, and PAGE#15) as in the pIEF experiment described in the previous section (Supporting Information Fig. 14). We assigned each parallel slice as a PAGE replicate (e.g. PAGE#05-r1, PAGE#05-r2, and PAGE#05-r3, etc.). First, we wanted to determine whether pIEF-LC-MS/MS analysis of peptides from replicate PAGE slices is reproducible. Therefore, we examined the distribution and numbers of identified peptides across the IPG strip in the PAGE-pIEF-LC-MS/MS approach. Similarly to the results described in the previous section, we observed a matching distribution of the identified peptides across the IPG strip from the pIEF analysis of replicate PAGE slices (Fig. 4), showing that the pIEF-LC-MS/MS separation was reproducible. The only difference that we found was in the acidic end (slices 1–5) in the pIEF analyses of PAGE#05-r1 and PAGE#05-r2 (Fig. 4A). We contributed this to improper rehydration at the acidic end of the IPG strip and underperformance of the IEF separation. Despite showing different peptide distribution, these five slices resulted in the identification of similar sets of peptides (data not shown). Next, we evaluated the overall reproducibility of GeLC-MS/MS and PAGE-pIEF-LC-MS/MS by looking at the numbers of identified proteins and peptides in a PAGE lane replicate. In both the GeLC-MS/MS and the PAGE-pIEF-LC-MS/MS approach there was a high consistency in the numbers of peptides and proteins identified (Table 2, Peptides, Proteins). The only exception was the pIEF analysis of PAGE#05 in which 6557, 7513, and 8569 peptides were identified in the three PAGE replicates. This difference was most probably a consequence of improper IPG strip rehydration, as discussed above. We further examined the overlap of protein identifications in all three PAGE replicates. As expected, in both experiments, the number of total proteins, i.e. those identified together in all three replicates, (Table 2, Total proteins) was higher compared with the number of identified proteins in a single replicate analysis (Table 2, Proteins). Meanwhile, the percentage of the total proteins that were also identified across all three replicates was higher for GeLC-MS/MS (65–67%) than for PAGE-pIEF-LC-MS/MS (52–63%). This showed that PAGE-pIEF-LC-MS/MS was slightly less reproducible than GeLC-MS/MS. Finally, we examined the correlation of protein intensity across PAGE replicates (Table 2, correlation protein intensity). Overall GeLC-MS/MS showed higher Pearson’s correlation values ranging from 0.75 to 0.98, compared with PAGE-pIEF-LC-MS/MS with values between 0.67 and 0.97, further supporting the evidence that PAGE-pIEF-LC-MS/MS was less reproducible than GeLC-MS/MS. Nevertheless, we observed that in both approaches, for all PAGE slices, two out of the three replicates showed very high correlation coefficients of the protein intensity: PAGE-r2/PAGE-r3 in GeLC-MS/MS and PAGE-r3/PAGE-r1 (slices #05 and #10) and PAGE-r1/PAGE-r2 and PAGE-r2/PAGE-r3 (slice #15) in PAGE-pIEF-LC-MS/MS (Table 2). These results indicated that very high reproducibility of PAGE-pIEF-LC-MS/MS can indeed be achieved.
Affiliation: Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.